1. David McFall

2. How caffeine interacts with the body  The binding of the hormone adenosine to an adenosine receptor in the brain brings about sleep.  Caffeine blocks adenosine from being connected to the adenosine receptor.  The hormone epinephrine is then produced. This creates the notable effects of caffeine.

3. Coffee Coffee has a high caffeine content due to its high concentration in its endosperm. Coffee can be decaffeinated by a treatment of methylene chloride.

4. The Effects of Coffee  Coffee’s side effects are drawn from the release of the hormone epinephrine, shown here. The most notable effects include:  high heart beat  high blood pressure  higher blood sugar content  tense muscles Epinephrine

5. 20 mg of Caffeine per ounce0.6 to 1.4 mg of Caffeine per ounce Starbucks X-bold Sumatra Roast (Coffee A) Starbucks Decaf House Blend (Coffee B)

6. Escherichia coli  The model for this experiment is Escherichia coli (E. coli).  It is found in the intestinal tract of most animals, including humans.  Most importantly, E. coli has been tested in conjunction with caffeine before in previous studies.

7.  A study from the Lithuanian State Science and Studies Foundation concluded that when in high concentration, caffeine lowers the population of E. coli.  Another study from the Indiana University School of Medicine supported the previous study.

8.  To determine the effect of coffee on human coli form populations.

9. Null Hypothesis: Neither caffeinated nor decaffeinated coffee will have an effect on the population of E. coli at any concentration. Alternative Hypothesis: Caffeinated and decaffeinated coffee will significantly effect survivorship of E. coli

10.  E. coli  Permanent Marker  Latex Gloves  Micropipettes  Microtubes  Caffeinated Coffee  Decaffeinated Coffee  Sterile Distillation Fluid  58 LB agar plates (Per Liter:1% Tryptone, 0.5% Yeast Extract, and 1% NaCl)  LB media  Klett Spectrophotometer  0.22 micron syringe filters  2 Sterile Filters  Bunsen Burners  Spread Bar  Incubator  Ethanol  Matches  Vortex

11. 1.E. coli was grown overnight in sterile LB media. 2.A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3.The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8 cells/mL. 4.The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL. 5.Caffeinated and decaffeinated coffee was mixed with the appropriate amount of SDFto create coffee concentrations of 10%, 1%, and 0.1%. 6.100 µL of cell culture was then added to the solutions, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 7.The solutions were vortexed and allowed to sit at room temperature for 15 minutes. 8.100 µL aliquots were removed from the tubes and spread on LB plates. 9.The plates were incubated at 37 degrees for 24 hours. 10.The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell. 11.The appropriate statistical analyses were performed to adequately assess the hypothesis.

12. 1.Repeat steps 1-6 in the Preliminary Procedure. 2.Both brands of coffee were brewed as directed. 3.The coffee was then sterile filtered. 4.200 µL of sterile coffee were removed from the tubes and spread on LB plates. 5.The plates were incubated at 37 degrees for 24 hours. 6.100uL of bacteria (control tube) was spread onto each plate. 7.The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell. 8.The appropriate statistical analyses were performed to adequately assess the hypothesis.

13. Table 1Control (x2).01x (x4).1x (x4)x (x4) E. coli.1 ml Sterile Distillation Fluid9.9 ml9.89 ml9.8 ml8.9 ml Coffee0 ml.01 ml.1 ml1 ml Total Volume10 ml Chart of Concentration

14. P>0.05

15. T Critical= 3.49 (Significant) Variable Concentration T Value Interpretation.01x CAFF2.2 Insignificant.1x CAFF3.46 Insignificant x CAFF4.78 Significant.01x DCAFF7.63 Significant.1x DCAFF4.54 Significant x DCAFF3.69 Significant

16.  Caffeinated coffee significantly increased the population of E. coli, but only in the 10% concentration.  Decaffeinated coffee significantly increased the population of E. coli in all concentrations.

17.  Test more brands of coffee  Use a different model  Test coffee in conjunction with other caffeinated beverages, such as tea or energy drinks

18.  Plating was not exactly synchronized, which could have resulted in extra time for bacterial replication. A team of students could remedy this technical problem.